Organophosphate Ester, 2-Ethylhexyl Diphenyl Phosphate (EHDPP

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Ecotoxicology and Human Environmental Health

Organophosphate Ester, 2-Ethylhexyl Diphenyl Phosphate (EHDPP), Elicits Cytotoxic and Transcriptomic Effects in Chicken Embryonic Hepatocytes and its Biotransformation Profile Compared to Humans Jinyou Shen, Yayun Zhang, Nanyang Yu, Doug Crump, Jianhua Li, Huijun Su, Robert J. Letcher, and Guanyong Su Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06246 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

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Environmental Science & Technology

Table of Content (TOC) Graphical Art

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Organophosphate Ester, 2-Ethylhexyl Diphenyl Phosphate (EHDPP), Elicits Cytotoxic and

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Transcriptomic Effects in Chicken Embryonic Hepatocytes and its Biotransformation Profile

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Compared to Humans

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Jinyou Shen†,#, Yayun Zhang†,#, Nanyang Yu‡, Doug Crump§, Jianhua Li†, Huijun Su†, Robert J. Letcher§,

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Guanyong Su†,*

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† Jiangsu

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Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, P. R. China

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‡

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University, Nanjing 210023, China

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§

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Research Centre, Carleton University, Ottawa, ON, K1A 0H3, Canada

Key Laboratory of Chemical Pollution Control and Resources Reuse, School of Environmental and

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing

Ecotoxicology and Wildlife Health Division, Environment and Climate Change Canada, National Wildlife

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* Corresponding authors:

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Tel.: 86-1-395-176-3661,

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E-mail: [email protected],

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#

These two authors contribute this work equally.

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Abstract The effects of 2-ethylhexyl diphenyl phosphate (EHDPP) on cytotoxicity and mRNA expression, as

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well as its metabolism, were investigated using a chicken embryonic hepatocyte (CEH) assay. After

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incubation for 36 h, the lethal concentration 50 (LC50) was 50 ± 11 μM, suggesting that EHDPP is one of a

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small cohort of highly toxic organophosphate esters (OPEs). By use of a ToxChip polymerase chain reaction

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(PCR) array, we report modulation of 6, 11 or 16/43 genes in CEH following exposure to 0.1, 1 or 10 μM

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EHDPP, respectively. The altered genes were from all 9 biological pathways represented on the ToxChip

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including bile acids/cholesterol regulation, glucose metabolism, lipid homeostasis and the thyroid hormone

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pathway. After incubation for 36 h, 92.5 % of EHDPP was transformed, and one of its presumed

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metabolites, diphenyl phosphate (DPHP), only accounted for 12% of the original EHDPP concentration.

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Further screening by use of high-resolution spectrometry revealed a novel EHDPP metabolite, hydroxylated

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2-ethylhexyl monophenyl phosphate (OH-EHMPP), which was also detected in a human blood pool.

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Additional EHDPP metabolites detected in the human blood pool included EHMPP and DPHP. Overall, this

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study provided novel information regarding the toxicity of EHDPP and identified a potential EHDPP

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metabolite, OH-EHMPP, in both avian species and humans.

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Keywords: organophosphate ester, 2-ethylhexyl diphenyl phosphate (EHDPP), lipid homeostasis, avian

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species, humans

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Introduction

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The production and use of brominated flame retardants (BFRs) such as polybrominated diphenyl ethers

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(PBDEs) and hexabromocyclododecane (HBCDD) has been restricted under the Stockholm Convention. As

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a result, the market share of organophosphate ester (OPE) flame retardants (FRs) has been increasing

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significantly in recent years.1 In Europe, the total consumption of OPE-FRs in 2006 was approximately 90,000

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tonnes (equivalent to 20% of the total consumption) and the current market share of OPE-FRs is even greater.2

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In China, the annual consumption of OPE-FRs was approximately 70,000 tons in 2007 with an annual

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estimated increase of 15 %.3 Thus, there is great concern regarding the occurrence, transformation and health

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effects of OPE-FRs in the environment.4, 5

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2-Ethylhexyl diphenyl phosphate (EHDPP) is one typical OPE with a phosphate ester backbone

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structure.2 EHDPP is used widely in various products including, polyvinyl chloride (PVC), rubber, and food

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packaging, as a plasticizer and a FR.6,

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products and therefore can easily leach out into the environment during manufacture, usage, and disposal of

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products.8

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As an additive to polymers, EHDPP is not chemically bound to

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EHDPP is ubiquitous in various environmental samples, such as indoor air, dust, surface water, wildlife

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and even humans.8-12 For instance, in indoor dust from Portugal, Denmark, the U.K., and the U.S., EHDPP

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was a dominant contaminant, with concentrations up to 1.1 μg/g, 0.79 μg/g, 1.6 μg/g, and 3.3 μg/g,

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respectively.13-15 Kim et al investigated the OPEs in a wastewater treatment plant in New York, and found

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that EHDPP was one of the dominant compounds in ash samples with a concentration of 288 ng/g dry weight.10

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Two recent studies have reported detection frequencies of EHDPP in surface water samples from Taihu Lake

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in China were 88 % and 100 % with mean concentrations of 1.9 ng/L and 2.8 ng/L, respectively, which could

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pose a risk to local aquatic organisms.16, 17 Notably, EHDPP concentrations in biota were higher than in surface

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water from the same sampling sites. In a Swedish lake and Manila Bay (Philippines), concentrations of 14 and

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2.1 μg EHDPP/g lipid weight in fishes were reported, respectively.18, ACS Paragon Plus Environment

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In a recent study, Zhao et al

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investigated human prenatal exposure to OPEs by analyzing chorionic villus samples; EHDPP was detectable

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in 100% of analyzed samples with a median concentration of 13.6 ng/g dry weight.9 These extremely high

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detection frequencies warrant a comprehensive assessment of potential adverse effects to exposed organisms.

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Data regarding the toxicity of EHDPP are scarce, but a few studies have shown that EHDPP can induce

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adverse effects such as endocrine disruption and developmental and neurotoxic effects in exposed

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organisms.20-22 Specifically, recent studies have investigated the activation of human peroxisome proliferator-

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activated receptor gamma (PPARγ) and estrogen-related receptor γ (ERRγ) using luciferase reporter gene

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assays and demonstrated that EHDPP bound to and activated PPARγ and ERRγ.20, 21 Schang et al investigated

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the possible activity of EHDPP as an endocrine disruptor in MA-10 mouse Leydig tumor cells, and reported

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that EHDPP altered the expression of genes involved in mitochondrial activity, cell survival, superoxide

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production, and basal or stimulated steroid secretion.22 Behl et al evaluated a set of OPEs, including EHDPP,

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using multiple cell-based in vitro assays and demonstrated that EHDPP (1-10 μM) exhibited comparable

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developmental and neurotoxic effects as two BFRs, 3,3′,5,5′-tetrabromobisphenol A (TBBPA) and 2,2′4,4′-

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brominated diphenyl ether (BDE-47).23 Overall, EHDPP toxicity data are still unavailable for most organisms,

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including avian species, and more comprehensive studies are warranted to understand its underlying

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mechanism(s) of toxicity.

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In terms of biological transformation of EHDPP, an in vitro study using human liver microsomes

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suggested two crucial pathways: 1) hydroxylation of the phenyl ring; and 2) O-dealkylation between the 2-

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ethylhexyl functional groups and phosphate acid. These pathways would result in the formation of diphenyl

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phosphate (DPHP) and hydroxylated EHDPP (OH-EHDPP).24 DPHP has been frequently reported in human

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urine samples and its concentrations were positively correlated with thyroid hormones (T4).25-27 However,

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DPHP is not a specific metabolite for EHDPP because it can be derived from multiple parent compounds such

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as triphenyl phosphate (TPHP), resorcinol bis(diphenylphosphate) (RDP), and isopropylated and tertbutylated

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triarylphosphate esters (ITPs & TBPPs).28-31 OH-EHDPP appears to be an unique biomarker of EHDPP, but ACS Paragon Plus Environment


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concentrations of OH-EHDPP in human samples are very low (i.e. 0.09 ng/mL urine).32 Investigating the

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metabolism of EHDPP and identifying potential biomarkers is critical for understanding its adverse effects to

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organisms. Currently, information on the metabolism of EHDPP remains unknown for avian species, and

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differences of biotransformation between avian species and humans are also unknown.

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The present study investigated the cytotoxicity and mRNA expression levels of 43 genes from 9 pathways

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– bile acids/cholesterol regulation, cell cycle, DNA repair, glucose metabolism, immune response, oxidative

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stress, the thyroid hormone pathway, lipid homeostasis, and xenobiotic metabolism – in chicken (Gallus gallus

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domesticus) embryonic hepatocytes (CEHs) following exposure to EHDPP. Additionally, the concentration

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of EHDPP and DPHP in cultured cells and medium were quantified to characterize the mass balance between

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EHDPP and DPHP and cell accumulation. Finally, other potential metabolites besides DPHP were identified

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by use of high-resolution quadrupole-time of flight mass spectrometry (Q-ToF-MS), and newly identified

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metabolites were further screened in a pool of human blood from the Chinese population to examine the

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difference of EHDPP metabolism between an avian in vitro assay (CEHs) and humans.

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Experimental section

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Chemicals

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All chemicals, i.e. EHDPP (CAS No.: 1241-94-7), DPHP (CAS No.: 838-85-7), and tris(1,3-dichloro-2-

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propyl) phosphate (TDCIPP; CAS No.: 13674-87-8), were purchased from Sigma-Aldrich (St. Louis, MO,

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U.S.A.). The purity of these chemicals was reported to be greater than 99% by manufacturer.

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Preparation of Chicken Embryonic Hepatocytes (CEH)

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Detailed information for the preparation of CEHs can be found elsewhere.33, 34 In brief, we purchased

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fertilized and unincubated white leghorn chicken eggs from the Canadian Food Inspection Agency , and these

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eggs were incubated (37.5 °C, 60 % relative humidity) for 19 days. After incubation for 19 days, embryos ACS Paragon Plus Environment

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were decapitated and livers were removed and pooled rapidly. For cell separation and reduction of cell

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aggregation respectively, hepatocytes were treated with Percoll and DNase I in sequence. After centrifugation,

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cells were then suspended by use of 32 mL of Medium 199 that were supplemented with 1μg/mL of insulin

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and thyroxine. Once the cells were distributed into each well of 48-well plate, the hepatocytes were incubated

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for 24 h under conditions of 37.5 °C and 5% CO2. After 24 h, the CEHs were dosed to DMSO (2.5 μL/well;

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n = 3) and EHDPP (nominal concentrations: 100, 20, 10, 2, 1, 0.1 μM for cell viability; 10, 1, and 0.1 μM for

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variation of mRNA levels; 1 μM for in vitro metabolism; n = 3 for all treatments) and incubated for additional

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hours (i.e. 0, 12 or 36 h) depending specific purposes.

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Cell viability

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ViaLightTM Plus BioAssay kit was used for the measurement of adenosine triphosphate (ATP) in CEHs

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exposed to EHDPP for 36h. Based on our previous studies, TDCIPP at a nominal concentration of 300 μM

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was used as a positive control.30, 35 Following the 36h incubation period, the plates were maintained at 20 oC

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for no less than 5 min. Then, CEH medium was gently aspirated from each well of the plates, and fresh medium

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and cell lysis reagent was successively added into each well. After mixed well, a 100 μL aliquot of this

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medium/lysis reagent mixture was transferred to a luminometer plate, and incubated for 2 min at 20 oC. The

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mixture was then mixed with an aliquot of 100 μL of ATP monitoring reagent plus, and immediately measured

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for intensity of luciferase luminescence by use of the Luminoskan Ascent.

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Variation of mRNA levels in CEH following exposure to EHDPP

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EHDPP was investigated at three concentrations (10, 1 and 0.1 μM) to determine effects on gene

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expression. After exposure for 36 h, we further examined variation of mRNA levels in CEH by use of the 4th

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generation custom chicken RT2 Profiler PCR Array (ToxChip) built in a 96-well plate.34 The 96-well ToxChip

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contained 43 target genes and 5 control genes in duplicate. Biological functions of these 43 target genes were ACS Paragon Plus Environment


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described in Table S1, and the 5 control genes involved 1 positive PCR control (PPC), 1 reverse transcription

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control (RTC), 2 housekeeping genes, and 1 genomic DNA control, respectively.

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Isolation of total RNA from CEH were detailed in our previous publications.30, 35 In brief, plates were

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removed from the -80 oC freezer, and the cell lysis reagent was added into each well of plates. Then, 200 ng

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of RNA was reverse-transcribed to complimentary DNA (cDNA), that was gently mixed with the RT2 SYBR

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Green Mastermix (Qiagen). A 25 μL aliquot of this mixture was immediately added to each well on the 96-

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well ToxChip. All arrays were measured in the Agilent Stratagene MX3005 (Agilent Technologies, Santa

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Clara, CA, U.S.A.) using the thermal profiles reported in our previous publications.30,

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measurement of PCR arrays, amplification of gene expression was not observed for the genomic DNA

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contamination control (i.e. Ct value ≥ 35), and PPC and RTC controls met the appropriate QA/QC criteria (i.e.

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ΔCt ≤ 5; ΔCt = Ct values of RTC – Ct values of PPC). These results ensure robust gene expression analysis.

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Importantly, the relative cycle thresholds of the two housekeeping genes were invariable (ANOVA, p > 0.05)

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regardless of treatment with EHDPP at any of the administered concentrations.

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We During the

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In Vitro Metabolism of EHDPP

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A non-cytotoxic concentration of EHDPP (1 μM; see Cell Viability section) was administered to CEH

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for the in vitro metabolism evaluation. DMSO-treated cells were used as experimental blanks. After incubation

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for 0, 12, and 36 h, culture medium in each of wells was transferred to amber glass vials. For comparison on

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EHDPP amounts between CEH cell and medium sample, the remaining cell layer in each of wells was also

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collected by washing twice with 100 μL of ethanol, and transferred into amber glass vials. Prior to chemical

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analysis, the collected medium or the ethanol/cell mixture was diluted by 25 times with fresh methanol. Then,

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a 1000 µL aliquot of diluted samples were spiked with 20 ng of d15-TPHP (as internal standard for

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quantification of EHDPP) and d10-DPHP (as internal standard for quantification of DPHP), filter-centrifuged,

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and ready for instrumental analysis. ACS Paragon Plus Environment

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Extraction of EHDPP Metabolites from Blood Samples

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Screening of EHDPP metabolites was conducted in a single human blood pool comprising blood samples

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collected during routine physical examinations of n = 99 people (50 males and 49 females) from Jiangsu

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Province (cities of Suzhou, Taizhou, Yangzhou and Nanjing), eastern China. The age of recruited people

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ranged from 18 to 87 years old. This research was approved by the research ethics boards of involved

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universities, and the informed consent process has been conducted for all the volunteers prior to blood

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sampling. An aliquot of 100 μL was taken from each of the 99 samples, and pooled together for further

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extraction. Prior to extraction, 0.5 mL of blood sample was transferred into a disposable plain conical

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centrifuge tube, and spiked with 10 ng of d15-TPHP and d10-DPHP. After that, 1.5 g Na2SO4, 0.1 g NaCl and

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2 mL acetonitrile containing 1 % acetic acid were added to the sample. The sample tube was vortexed for 20

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s and centrifuged for 5 min at 3500 rpm. A total volume of 1.5 mL of the supernatant fraction was transferred

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into a disposable plain conical centrifuge tube, and 2.0 g Na2SO4 was added to the tube. The tube was vortexed

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and centrifuged as described above. An aliquot of 1.0 mL of the supernatant fraction was transferred into a

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new tube. The extract was evaporated under a stream of nitrogen to dryness, and 0.2 mL of methanol was

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added to the sample. The sample was vortexed and was then ready for injection into the analytical instruments.

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During the extraction process, recoveries of d10-DPHP and d15-TPHP were calculated to be 87 ± 8% and 93 ±

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7% by comparing the instrumental differences of the spiked extracts and pure standards, respectively,

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indicating a qualitative extraction of EHDPP metabolites from the plasma samples.

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Liquid Chromatography-Quadrupole Mass Spectrometry (LC-MS/MS)

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LC-ESI-MS/MS was used for: 1) the accurate quantification of EHDPP and DPHP in medium and cell

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samples; and 2) investigation of daughter ions of newly detected EHDPP metabolites. For more details

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regarding analysis of EHDPP or DPHP, please refer to our previous publications.5, 25 In brief, analysis of these ACS Paragon Plus Environment


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three compounds has been conducted by use of a high performance liquid chromatography (LC) coupled to a

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tandem quadrupole mass spectrometer (MS/MS) (Waters, Milford, MA, U.S.A.). Chemicals were separated

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by a C18 column (Waters Symmetry, 100 × 2.1 mm, particle size: 3.5 μm). The LC mobile phases were water

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(containing 2 mM of ammonium acetate (AA)) and methanol (containing 2 mM of AA) with a constant flow

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rate of 0.5 mL/min. The gradient of mobile phase was set as: 0 min, 5% methanol (containing 2 mM of AA);

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0−5 min, 95% methanol (containing 2 mM of AA); hold for 1 min; 6−6.1 min, 5% methanol (containing 2

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mM of AA) and hold for 4.9 min. For detection of EHDPP, the electrospray ionization (ESI) source was

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operated in multiple reaction monitoring (MRM) mode and in positive mode, whereas for DPHP the ESI

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source was operated in MRM mode and in negative mode. For OH-EHMPP metabolite, the ESI source was

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operated in daughter ion mode and in negative mode to investigate formed daughter ions from potential OH-

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EHMPP metabolites.

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Liquid Chromatography-Quadrupole-Time of Flight-Mass Spectrometry (LC-Q-ToF-MS)

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LC-Q-ToF/MS was used for screening potential EHDPP metabolites in CEHs and human blood samples.

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The LC-Q-ToF/MS is an Agilent 1200 LC system that was coupled with an 6520A Q-ToF-MS system (Agilent

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Technologies, Mississauga, ON, Canada). The Phenomenex (Torrance, CA, U.S.A.) Luna C18 column (2.0 ×

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50 mm, particle size: 3 μm) has been used for separation of target compounds. The LC mobile phases were

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water (containing 2 mM of ammonium acetate (AA)) and methanol (containing 2 mM of AA) with a constant

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flow rate of 0.5 mL/min. The gradient for mobile phases was set as: 0 min, 5 % methanol (containing 2 mM

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of AA); 0−5 min, 95 % methanol (containing 2 mM of AA); and hold for 10 min; and finally with a post run

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time of 15 min. The LC-Q-ToF-MS was equipped with an ESI source, and each of the samples were injected

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twice with ESI operated in either positive or negative mode. Before instrumental analysis, the high resolution

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Q-ToF system was tuned and calibrated. Specifically, the resolution of Q-ToF system was >20,000 at m/z

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322.0481 (Δ = 0.01 ppm) or m/z 301.9981 (Δ = 0.15 ppm) when the ESI interface was operated in positive or ACS Paragon Plus Environment

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negative mode, respectively. For negative ESI, the reagent containing TFA anion (theoretical m/z 112.9855)

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and HP-0921 formate adduct (theoretical m/z 966.0007) were consistently introduced into the Q-ToF-MS as

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reference masses. For positive ESI, purine (theoretical m/z 121.0509) and HP-0921 (theoretical m/z 922.0098)

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were used as reference masses.

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Data Analysis

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For cell viability, luciferase activity for each sample was adjusted for background (300 μM TDCIPP)

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prior to the normalization as compared to the solvent control (DMSO). By use of GraphPad 5 (San Diego,

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CA, U.S.A.), a nonlinear regression curve has been fitted for visualization of cell viability of EHDPP. PCR

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array data were analyzed by use of the MxPro software (v4.10, Agilent Technologies, Santa Clara, CA,

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U.S.A.), and the Ct was set to 0.1. A classic 2−ΔΔCt method has been used for calculation of fold change mRNA

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expression compared to the vehicle control.36 Then, an one-way ANOVA (p < 0.05) analysis was used for

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investigation of differences in fold change as compared to DMSO. For visualization of gene expression, target

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genes with nonsignificant fold-changes (i.e. p > 0.05) or those less than 2-fold were set to 0 to minimize noise.

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Finally, gene clustering and heatmaps were generated by use of a “gplots” package in R software (version:

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3.0.2). For concentrations of EHDPP and DPHP in CEH, the peak in the chromatogram with an S/N ratio of

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10 and 3 was identified as the method limit of quantification (MLOQ) and method limit of detection (MLOD),

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respectively. MLOQ and MLOD for EHDPP were 0.03 and 0.01 ng/mL, respectively. MLOQs and MLODs

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for DPHP were 0.6 and 0.2 ng/mL, respectively. Concentrations lower than MLODs, and those between

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MLOQs and MLODs were reported as not detected (ND) and < MLOQ, respectively.

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Results and discussion

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Cytotoxic Effect Following Exposure to EHDPP

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Cell viability was evaluated by measuring the cytoplasmic ATP levels in CEH exposed to a wide range ACS Paragon Plus Environment


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of EHDPP concentrations (0.1 μM to 100 μM). Any form of cell damage/death caused by EHDPP would be

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reflected by lower concentrations of cytoplasmic ATP.37 A statistically significant decrease in the viability

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(ANOVA, p < 0.05) was observed at exposure concentrations ≥ 10 μM EHDPP. Based on the fitted curve

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(Figure 1A), the lethal concentration 50 (LC50) was calculated to be 50 ± 11 μM. To our knowledge, this is

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the first report regarding cytotoxic effects of EHDPP in CEH. In a recent study, Hu et al investigated the

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cytotoxicity of EHDPP following exposure to the human placental choriocarcinoma cell line, JEG-3. The

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authors did not observe any effects on cell viability or proliferation at concentrations ranging from 5 to 40

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μM.20 The observed differences might be due to the different cell lines used or species-specific responses.

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Cell death was identified as a useful endpoint to prioritize chemical contaminants in vitro, and to date,

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the CEH assay has been used to evaluate the cytotoxicity of 19 OPEs (Table S2).30, 33-35, 38 Including the present

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study with EHDPP, of the 20 OPEs evaluated, 13 did not decrease cell viability up to the highest exposure

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concentration. Alternatively, significant decreases in viability were observed for the following 6 OPEs as well

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as EHDPP (50 ± 11 μM): tris(1,3-dichloro-2-propyl) phosphate (TDCIPP, LC50: 60.3 ± 45.8 μM), tris(2-

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butoxyethyl) phosphate (TBOEP, 61.7 ± 43 μM), triphenyl phosphate (TPHP, 47 ± 8 μM), bis(2-ethylhexyl)

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phosphate (DEHP, 40.6 ± 13 μM), p-tert-butylphenyl diphenyl phosphate (BPDP, 148 ± 83 μM), and

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isopropylphenyl phosphate (IPPP, 179 ± 100 μM).26, 28-30, 33 Taken together, the LC50 of EHDPP is comparable

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to a small cohort of highly cytotoxic OPEs, i.e. DEHP, TPHP, TDCIPP and TBOEP (Figure 1B), indicating

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that EHDPP should be prioritized for further evaluation.

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Alteration of mRNA Expression in CEH Exposed to EHDPP

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The mRNA expression levels of 43 genes associated with the 9 pathways represented on the ToxChip

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were examined in CEH following exposure to EHDPP at concentrations of 0.1, 1 or 10 μM. At least one gene

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was altered in each of the pathways (Figure 2, Table S3) and 6, 11, or 16/43 genes were significantly

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dysregulated following exposure to 0.1, 1, and 10 μM EHDPP, respectively. The greater transcriptomic ACS Paragon Plus Environment

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response observed in CEHs treated with 10 μM demonstrated a concentration-dependent effect on gene

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expression; individual fold-change values for those genes dysregulated by all treatment groups also followed

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that pattern (Figure 2, Table S3).

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All three genes from the ToxChip associated with bile acids/cholesterol regulation (CYP7B1, FGF19 and

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NR5A2) were dysregulated by EHDPP; CYP7B1 and FGF19 were down-regulated and NR5A2 was up-

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regulated. These genes play an important role in digestion and absorption of lipids in the small intestine.39 As

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a cytochrome P450 enzyme associated with cholesterol synthesis, CYP7B1 catalyzes the conversion of 25-

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hydroxycholesterol and 27-hydroxycholesterol to bile acid intermediates,40 and its inactivation could cause a

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congenital bile acid synthesis deficiency in organisms.41 FGF19 , in combination with FGF receptor 4

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(FGFR4), involved with the regulation of bile acid biosynthesis, gallbladder filling, and glucose and lipid

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metabolism.42 NR5A2, also known as the liver receptor homolog-1 (LRH-1), plays a predominant role in

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development, reverse cholesterol transport and bile acid homeostasis.43 Its expression could activate the

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transcription of CYP7A1, which catalyzes the rate-limiting step in bile acid biosynthesis.44 Overall, genes

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associated with bile acids/cholesterol homeostasis were altered by EHDPP, highlighting a novel mechanism

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of action that should be further explored.

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Both genes associated with the glucose metabolism pathway (PDK4 and G6pc) were down-regulated

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following exposure to EHDPP. Glucose metabolism is critical for energy generation within the cell and the

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supply of fuel for respiration.45 PDK4 is associated with the conversion of glucose to fatty acid and its

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expression could be modulated by glucocorticoids (up-regulated) and insulin (down-regulated).46 PDK4 could

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decrease the conversion of glucose to acetyl-CoA, which is associated with energy generation and maintaining

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the balance between carbohydrate and lipid metabolism.47 G6pc is involved with maintenance of glucose

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homeostasis by gluconeogenesis and glycogenolysis and its transcription could lead to lower glucose levels

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by gluconeogenesis, which could be regulated by genes associated with energy homeostasis.48, 49 Collectively,

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down-regulation of these two genes could result in a glucose metabolism imbalance and this adverse effect ACS Paragon Plus Environment


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associated with EHDPP exposure requires further investigation.

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Two of the 4 genes associated with lipid homeostasis (ACSL5 and SLCO1A2) were significantly down-

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regulated in CEH following exposure to EHDPP. ACSL5 is a synthetase involved with the activation of fatty

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acids to acyl-CoAs and plays an anabolic role in lipid metabolism with high rates of triacylglycerol synthesis.50,

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51

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uptake of a wide range of drugs and xenobiotics.52 Additional substrates (e.g. bile acids, steroids, thyroid

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hormones and their conjugates) can also be transported by SLCO1A2.53 The concomitant down-regulation of

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ACSL5 and SLCO1A2 could imply a disruption of lipid metabolism and energy storage by EHDPP. Alteration

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of sphingolipid homeostasis by OPE-FRs was also reported in previous studies.54-56 Specifically, Du et al and

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Morris et al reported that TPHP could disturb lipid metabolism in vitro (i.e. zebrafish liver) or in vivo (i.e.

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mice), demonstrating a concordant endpoint in different model systems.55, 56 In a recent study, Zhao et al

292

reported that ln-transformed sphingolipid levels were negatively correlated with EHDPP concentrations in

293

human blood.54

SLCO1A2 encodes an organic anion-transporting polypeptide, which is an important mediator of cellular

294

Two of the 4 genes on the ToxChip associated with the thyroid hormone pathway (TTR and THRSP)

295

were down-regulated in a concentration-dependent manner following exposure to increasing concentrations

296

of EHDPP. The thyroid hormone pathway plays an important role in normal central nervous system

297

development in avian species, among many other critical roles.57 TTR is a major thyroid hormone carrier – in

298

addition to albumin – in avian species that transports thyroid hormones to target tissues.58 A depletion of

299

available thyroid hormones via decreased transport could lead to reduced total serum T4 levels.59-62 In addition

300

to EHDPP, other BFRs and OPEs (e.g. PBDEs, TBBPA, TPHP) have been identified as potent competitors

301

for thyroxine binding to TTR.9,

302

lipogenesis and maintenance of triacylglycerol and medium-chain fatty acid levels.63,64 To the best of our

303

knowledge, in vivo studies evaluating the effects of EHDPP on the thyroid hormone pathway are currently

304

unavailable. Given the importance of the thyroid hormone pathway for avian species, its disturbance following

65

THRSP is a crucial protein involved with thyroid hormone-mediated


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exposure to various FRs represents an important research gap to further explore; in particular, via in vivo

306

studies.

307

Finally, across the other biological pathways represented on the ToxChip, multiple genes were responsive

308

to EHDPP exposure and provided additional insight with regards to the mechanism(s) of toxicity. For example,

309

genes from the cell cycle (GADD45a and DDB2), immune response (LEAP2), oxidative stress (OGG1) and

310

xenobiotic metabolism (SULT1B1 and FGA) pathways were dysregulated. Detailed network/pathway analysis

311

might elucidate additional information regarding the observed perturbations in CEHs.

312 313

EHDPP Degradation versus DPHP Formation in CEHs

314

In vitro metabolism of EHDPP was investigated by determining concentrations of EHDPP and its

315

potential metabolite, DPHP, in both medium and CEHs. We observed a rapid degradation of EHDPP in

316

medium and cells (Table 1 and Figure 3). Specifically, total concentrations (including both medium and cell

317

samples) of EHDPP at 0, 12 and 36 h were 0.96 ± 0.11, 0.33 ± 0.023 and 0.072 ± 0.008 μM, respectively

318

(Table 1). Concomitantly, total concentrations of DPHP at the same time points were ND, 0.1 ± 0.005 and

319

0.11 ± 0.02 μM, respectively (Table 1). At 36 h, approximately 92.5 % of the original EHDPP was depleted;

320

however, DPHP concentrations were only approximately 12 % of the original EHDPP concentration (Figure

321

3). A previous CEH in vitro TPHP metabolism study reported similar patterns regarding TPHP degradation

322

and DPHP formation.30 Specifically, by use of the same in vitro CEH assay, Su et al observed rapid

323

degradation of TPHP (i.e. 98 % of TPHP was depleted by 36 h) and the resulting concentrations of DPHP

324

accounted for 17 % of the initial TPHP dosing concentration.30 Taken together, these results suggest: 1) both

325

EHDPP and TPHP are susceptible to rapid degradation in CEHs; and 2) DPHP represents only one of the

326

metabolites of EHDPP or TPHP.

327

Based on the connection between cellular accumulation and specific potency, cell enrichment of EHDPP

328

and DPHP were determined in this study. Concentrations of EHDPP in CEH were 0.57 ± 0.03, 0.19 ± 0.02 ACS Paragon Plus Environment


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and 0.042 ± 0.007 μM at time points of 0, 12 and 36 h, which represented 59 %, 57 % and 58 %, respectively,

330

of the total EHDPP concentration in wells. DPHP, on the other hand, was only detected in medium and not

331

CEHs at the two latter time points (12 and 36h). The Log octanol-water partition coefficient (Log KOW) of

332

EHDPP is 5.73 compared to 2.88 for DPHP, and therefore could help explain the preferential bioaccumulation

333

and prominent effects (e.g. cell viability and gene expression) observed for EHDPP.2, 66 Overall, the variable

334

distribution of EHDPP and DPHP in medium and cells indicated that: a) EHDPP was taken up by CEH; b)

335

DPHP was formed as a metabolite of EHDPP; and c) the metabolite, DPHP, was eliminated completely from

336

cells into the medium. These assumptions are consistent with the detection of DPHP in human fluids (i.e.,

337

urine) in previous studies.67, 68

338

DPHP has typically been regarded as a biomarker for measuring TPHP exposure in humans with frequent

339

detection in human urine samples from various countries around the world.25,

340

publications are raising questions regarding this link because: 1) DPHP can be formed from multiple parent

341

compounds (e.g. TPHP, EHDPP, RDP, ITP isomers, or TBPP isomers); and 2) a limited amount

342

(approximately 20 % or less) of DPHP was formed from TPHP via various in vitro assays that used human

343

microsomes, herring gull microsomes, CEHs or human serum.28, 30, 31, 70, 71 The results of the present study

344

provide additional evidence that DPHP is not a specific biomarker of TPHP given its detection in culture

345

medium following EHDPP exposure. Therefore, caution regarding the interpretation of DPHP detection is

346

recommended.

68, 69

However, recent

347 348

Comparison of EHDPP Metabolism in CEHs and humans

349

Previous in vitro studies, using human liver microsomes or S9 fractions, demonstrated that

350

biotransformation of OPEs can generate a wide array of metabolites formed by oxidative metabolism.70

351

Typically, microsomal enzymes (phase I) cause hydroxylation, O-dealkylation, oxidative diarylation and

352

reductive and oxidative dehalogenation, whereas S9 fractions (phase II) typically induce glucuronidation and ACS Paragon Plus Environment

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sulfation of metabolites generated during phase I metabolism.70, 72-74 On this basic knowledge, we created a

354

database that includes 19 potential EHDPP metabolites, all of which were further screened in the EHDPP-

355

exposed CEH samples by high resolution mass spectrometry (Table S4).

356

In addition to DPHP, we detected an ion of m/z 301.1214 in CEHs, which was absent in the blanks (Figure 4).

357

The m/z 301.1214 was presumed to be hydroxylated 2-ethylhexyl monophenyl phosphate (OH-EHMPP) with

358

a theoretical formula of C14H23O5P1 with [M-H]- of m/z 301.1210. Using the MS/MS mode of a UPLC-ESI-

359

TQ-S/MS operated in negative mode, we further examined the fragmentation of m/z 301, which can generate

360

multiple daughter ions including m/z 79, m/z 93 and m/z 173. We presumed that the m/z 79 daughter ion was

361

generated from a resonance stabilization of m/z 301 via a McLafferty rearrangement. The m/z 79 ion is a

362

phosphite anion ([PO3]-), which has been reported for other diesters, i.e. DEHP and DNBP, in previous

363

studies.25, 75 The daughter ions m/z 93 and m/z 173 were presumed to be phenolate anion ([C6H5O1]-) and

364

mono-phenyl phosphate anion ([C6H6P1O4]-), respectively, which are typical daughter ions for aryl phosphate

365

esters, i.e. DPHP.75 Collectively, our datasets based on both high resolution Q-ToF/MS and MS/MS suggested

366

that: 1) the newly detected ion could be OH-EHMPP; and 2) hydroxylation likely occurred on the functional

367

group of 2-ethylhexyl. However, due to limited authentic and pure analytical standards, accurate and thorough

368

identification and quantitation of this new metabolite was not possible at present. To the best of our knowledge,

369

this is the first report describing the detection of OH-EHMPP in a CEH metabolism study. In previous studies,

370

the alkyl chain of EHDPP tended to undergo oxidation as opposed to the phenyl rings in human liver

371

microsomes.24 In contrast, the presence of OH-EHMPP revealed a hydroxylation metabolism pathway for

372

EHDPP, and similar hydroxylation metabolism has been shown for flame retardants including TPHP and

373

HBCDD.66, 76, 77

374

To further elucidate the differences of EHDPP metabolism in CEH versus human samples, the 19

375

potential EHDPP metabolites were also screened in a human blood pool collected from individuals from

376

Jiangsu Province, eastern China. Three metabolites, including DPHP, OH-EHMPP and EHMPP, were ACS Paragon Plus Environment


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detected in the human blood pool and EHMPP showed the greatest instrumental intensity (Figure 5). These

378

results suggested that: 1) the formation of OH-EHMPP from EHDPP could occur in both humans and avian

379

species; and 2) differences exist between human and avian species because EHMPP was not detectable in

380

CEH medium but showed greatest intensity in human blood samples. The detection of EHMPP and DPHP in

381

human samples is well supported by several recent studies.32,

382

pooled human serum, van den Eede et al detected the formation of EHMPP with a minor amount of DPHP.71

383

By screening 14 OPE metabolites in urine samples of 128 elementary school-aged children, Araki et al

384

reported that EHMPP was detectable in 65 % of analyzed samples, and DPHP was detectable in 80 % of

385

analyzed samples with a mean concentration of 0.51 ng/mL.32 Currently, there is no available information

386

regarding DPHP or EHMPP concentrations in human blood.

387 388 389

Acknowledgements:

71

Specifically, by incubating EHDPP with

390

This research was supported by “Natural Science Foundation of Jiangsu Province” (Grant No.

391

BK20170830 and BK20180498), “the Fundamental Research Funds for the Central Universities” (Grant No.

392

30917011305), and “Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse (Nanjing

393

University of Science and Technology)” (Grant No. 30918014102). Dr. Su appreciates support from the

394

programs of “Thousand Talents Plan” and “Jiangsu Provincial Distinguished Professorship”.

395 396 397

Supporting Information The supporting information is available free of charge on the ACS Publications website. Further details

398

are given on biological function description of 43 target genes, a summary of LC50 values of 19 OPEs,

399

specific fold changes and p-values of 43 target genes following exposure to EHDPP, and a list of 19

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potential EHDPP metabolites.

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Table 1. Mean concentrations ± SD (units: μM) of 2-ethylhexyl diphenyl phosphate (EHDPP) and the metabolite diphenyl phosphate (DPHP) in cell culture medium and hepatocytes at three time points (0, 12 and 36 h) following exposure of chicken embryonic hepatocytes to EHDPP. Nominal concentration of EHDPP was 1 μM. Chemicals Samples 0h 12 h 36 h Medium 0.39 ± 0.08 0.14 ± 0.003 0.030 ± 0.001 EHDPP Cells 0.57 ± 0.03 0.19 ± 0.02 0.042 ± 0.007 % EHDPP in cells 59 % 57 % 58 % b Medium ND 0.10 ± 0.005 0.11 ± 0.02 DPHP Cells ND ND ND % DPHP in cells NCa NC NC a “NC” means not calculated because chemical was not detectable in cell or medium samples; b MLOQ and MLOD for EHDPP were 0.03 and 0.01 ng/mL, respectively. MLOQs and MLODs for DPHP were 0.6 and 0.2 ng/mL, respectively. Concentrations lower than MLODs and those between MLOQs and MLODs are reported as not detected (ND) and < MLOQ, respectively.



619 620 621 622 623

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Figure 1. Concentration-dependent cytotoxicity of 2-ethylhexyl diphenyl phosphate (EHDPP) in chicken embryonic hepatocytes (CEHs) incubated for 36h (A) and lethal concentration 50 (LC50) ranking for various organophosphate esters (OPEs) based on the CEH assay (B). The data in Figure 1A were log-transformed and the percent cell viability data (n=3 well per treatment group) were fit to a nonlinear regression curve (log (agonist) vs response) to determine the LC50 (±SEM). Error bars for each point represent the standard deviation of three replicates. LC50 values for a total of 19 OPEs are summarized in Table S2, and only those that exhibited cytotoxicity up to the highest administered concentrations are shown in Figure 1B.

624 625


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626 627 628 629 630


Figure 2. The relative mRNA expression profiles of 43 genes on the Avian ToxChip PCR array following exposure to 3 concentrations of 2-ethylhexyl diphenyl phosphate (EHDPP; 0.1 μM, 1 μM and 10 μM) for 36h. The heat map data represent the mean of three replicates. Genes with fold change lower than 2.0 and p > 0.05 were set to 0 to minimize noise. Green-black-red indicate down-, no-, and up-regulation. The detailed mRNA fold-change data are available in Table S3.

631 632



633 634 635 636 637

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Figure 3. Mass balance between 2-ethylhexyl diphenyl phosphate (EHDPP; the parent compound) and the metabolite, diphenyl phosphate (DPHP), following exposure of chicken embryonic hepatocytes (CEHs) to 1 μM EHDPP for 0, 12, and 36h. Reported concentrations (µM) were total concentrations of chemicals in both medium and cell samples. The data represent the mean of three replicate wells per time point.

638 639


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640 641 642 643 644


Figure 4. Detection of parent 2-ethylhexyl diphenyl phosphate (EHDPP) in culture medium at time point 0 h (A), diphenyl phosphate (DPHP) in culture medium at time point 36 h (B), and hydroxylated 2-ethylhexyl monophenyl phosphate (OH-EHMPP) in culture medium at time point 36 h (C) by use of liquid chromatography (LC)-electrospray (ESI)-time-of-flight mass spectrometry. Assignment of daughter ions (D) was generated from OH-EHMPP by use of LCESI-triple quadrupole mass spectrometry. For EHDPP, the ESI source was operated in positive mode, whereas the ESI source was operated in negative mode for DPHP and OH-EHMPP. “TM” means theoretical mass, and “ME” means error between theoretical and observed masses.

645 646



647 648 649 650

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Figure 5. Metabolites of 2-ethylhexyl diphenyl phosphate (EHDPP) detected in a pooled human blood sample by use of liquid chromatography-electrospray (ESI)-time-of-flight mass spectrometry: A) diphenyl phosphate (DPHP), B) hydroxylated 2-ethylhexyl monophenyl phosphate (OH-EHMPP), and C) 2ethylhexyl monophenyl phosphate (EHMPP). For all three metabolites, the ESI source was operated in negative mode. “TM” means theoretical mass, and “ME” means error between theoretical and observed masses.

651 652 653


Organophosphate Ester, 2-Ethylhexyl Diphenyl Phosphate (EHDPP

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